A New Method for Oblique Microscopical Illumination
BY
JOHN R. BAKER
{From the Department of Zoology and Comparative Anatomy,
Oxford)
With two Text-figures
T
HE purpose of the technique described in this paper is to provide the
easiest possible means of controlling the direction and degree of obliquity
of the light used to illuminate microscopical objects.
The method is applicable when striations or other regularly-repeated markings on objects are situated so close together that, with axial illumination, the
diffraction-spectrum they produce lies partly or wholly outside the back lens
of the objective. A clear image can in these circumstances only be produced
by throwing the direct light to one side of the back lens, and thus making
room for the spectrum on the other side. Methods for achieving this object
are familiar to microscopists. Advantages are claimed for the new method
described in this paper. The intention is to allow the microscope to be used
at its ultimate resolving-power with the greatest possible convenience and
effectiveness. The method is likely to be useful in the testing of high-power
objectives and in the study of the minutest markings on the shells of certain
diatoms.
The basis of the method is that the objective of the microscope is used as an
eyepiece through which the direction and degree of obliquity of the light are
examined while the mirror is tilted in various ways. The condenser has to be
in a special position in order to enable the objective to be used in this way.
No special apparatus is required. The source of light must be rather bright:
an ordinary ioo-watt filament-lamp with 'pearl' glass is very convenient.
('Opal' glass does not let through enough light.) Two stops with circular
apertures are needed, with some easy means of changing from one to the
other. One stop should have an aperture about 3 ! or 4 mm. in diameter, the
other about 15 mm. These will be called respectively the small and large
stops. The stop in use must be placed immediately in front of the lamp. (An
iris diaphragm can be used instead, but is rather less convenient.) The lamp
must be arranged in such a way that a line drawn from the centre of the mirror
through the centre of the stop would pass through the brightest part of the
lamp. It is also desirable that this line should be approximately at right angles
to the optical axis of the microscope. A stainless steel mirror is preferable to a
glass one, because it gives only a single image, but it is by no means a necessity
for the successful working of the method. An oil-immersion condenser of
wide aperture (such as Watson's Holoscopic) is required; it must be accurately
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234 Baker—A New Method for Oblique Microscopical Illumination
centred and the iris diaphragm below it must be kept wide open. An oilimmersion objective of high aperture is of course necessary. The eyepiece
used should be fairly powerful (I use a Watson's Holoscopic X 14). It is useful
to have also a short cork that fits the top of the draw-tube, with a hole about
8 mm. in diameter bored through the middle of it. The use of this will be
mentioned at the appropriate place.
The method for showing the striations of the shell of Amphipleura pellucida
will now be described as an example. As is well known, this elongated diatom
is transversely striated, the lines being exceedingly regular and closer together
than the shortest wave-length of visible light. It is best to have the diatoms
mounted in hyrax. Choose a specimen that lies somewhat apart from others
so as to avoid complications arising from the appearance of more than one
spectrum. (The presence of part of another diatom near the edge of the field
of view is not harmful.) It does not matter in what direction the chosen
diatom is orientated. In this description I shall suppose, for simplicity, that
it happens to lie with its long axis in the 'north and south' direction. The
striations, which will at first be invisible, will be directed east-west.
Place the small stop in front of the lamp and by means of the condenser
focus the image of the stop so that it is clearly seen while the outline of the
diatom is exactly in focus. (It is best to use a low-power eyepiece at this stage.)
The illumination is now 'critical' (see Text-fig. 1). Next remove the eyepiece
and substitute the bored cork. Put the large stop in front of the lamp, and
with one hand hold a sharp pencil with its point in the centre of the stop. Hold
the eye near the cork (the purpose of which is to make it easy to keep the line
of vision axial) and look down the tube of the microscope. Accommodate the
eye for distant vision. (Most microscopists do this reflexly when they look
through a microscope. If special glasses are used for distant vision, they should
be worn.) Now focus the condenser slowly upwards until the point of the pencil
is clearly seen. Stop focusing upwards directly this is achieved. If you go too
far, focus down again. The proper position has been achieved when a very
small movement of the condenser downwards would cause the pencil-point
to appear violently distorted and then vanish.
You have now secured what I call 'high-condenser illumination'. The
principle of it is illustrated in the central diagram of Text-fig. 1. The condenser having been raised above its so-called 'critical' position, the image of
the pencil-point has been brought above the diatom into the front focal plane
of the objective, which thus throws parallel rays from it up the tube. The eye,
being focused for distant vision, receives these rays and one sees the pencilpoint. The objective is now being used as an eyepiece.
Discard the pencil and put the small stop in front of the lamp. On looking
through the cork you will see the stop not filling the whole of the back lens of the
objective. We here deliberately use a stop that will not fill the back lens with
light. (To prevent any possibility of misunderstanding I mention here that
the iris diaphragm below the condenser must be fully open throughout the
whole procedure.)
Baker—A New Method for Oblique Microscopical Illumination
235
Keeping the eye at the hole in the cork, grasp the mirror in both hands and
turn it in its gimbals in such a way that the circular patch of light (the visual
image of the stop in front of the lamp) passes to one side of the back lens
of the objective. Stop when about half of the patch of light has passed out of
Eyepiece
objective
object
condenser
stop in front
of lamp
high- condenser
illumination
low- condenser
illumination
TEXT-FIG, I. Diagram showing the passage of rays of light in the microscope, with the
condenser in three different positions.
view and the other half remains at the edge of the back lens. (The circle of
light becomes distorted into an oval as it passes towards the edge.) Suppose
you have moved the light towards the east: now bring it slowly round the
edge of the back lens past the south-east towards the south. As it approaches
the south, a spectrum will be seen at the edge of the back lens of the objective
a little to the east of north (not to the west of north, as one might expect);
this spectrum will move towards the north and will be due north when the
direct light is due south. The violet end of the spectrum will be directed
236 Baker—A New Method for Oblique Microscopical Illumination
towards the centre of the back lens. Now tilt the mirror to and fro to bring
the light a short distance towards the middle of the back lens and back towards
the edge of the back lens again, keeping it in the north-south line. Meanwhile
watch the spectrum. Leave the mirror in the position that makes the spectrum
as bright and complete as possible. The more of the red end of the spectrum
you can bring within the back lens, the better. Text-fig. 2 shows the
appearance that should be obtained. (If a glass mirror be used, there will
be faint additional images of the stop, not quite coinciding with the bright
image.)
red end "I
violet end)
.
0fs
PeCbrum
edge of back lens o f
objective
dimly-lit part o f back
lens of objective
image of stop in front
o f lamp
TEXT-FIG. 2. Diagram showing the appearance seen on looking through the hole in the cork,
when the direction and degree of obliquity of the light are correct.
Remove the cork and place the high-power eyepiece in position. The striations of the diatom will now be clearly resolved. There is no need whatever
to use a colour-screen, even with a non-apochromatic objective. If perfection
is desired, focus carefully with the fine adjustment, replace the eyepiece by the
cork, and make very small movements of the mirror until the best possible
spectrum is obtained; then replace the eyepiece.
In whatever direction the diatom happens to be lying, the same procedure
should be adopted (with the obviously necessary changes).
Mr. W. E. Watson-Baker very kindly visited me in Oxford to see a demonstration of the method just described. He allows me to say that the striations
of Amphipleura pellucida were resolved more quickly than by any other method
he has ever seen, and that the images produced were superior to any obtainable by an oblique-light stop placed below the condenser. These comments
are mentioned in the hope that the opinion of such an experienced microscopist
will encourage others to give the method a trial.
It might be thought that the chromatic and other corrections of the objective
would be upset by placing the condenser above its 'critical' position. To
investigate this, I have made a careful examination of a number of species
of test-diatoms, using central (not oblique) high-condenser illumination. I
can find no evidence that the image is either better or worse than with
'critical' illumination. The expression 'critical' illumination is indeed a bad
one, for it begs the question whether this is better than other methods.
Hartridge (1919) has denied on both theoretical and practical grounds that
Baker—A New Method for Oblique Microscopical Illumination
237
'critical' is necessarily preferable to other kinds of illumination: it is simply an
easy and reliable way of getting good results.
If the substage iris diaphragm is somewhat closed and then kept in the same
position while the two methods of illumination are tried, a larger area of the
back lens of the objective will be filled with light by high-condenser than by
critical illumination (provided that a sufficiently large stop is used in front
of the lamp when the condenser is in the high position). When one is studying
diatoms such as Navicula lyra, which are not well seen under full-cone illumination, this fact must be kept in mind; for otherwise there will be a danger of
using too wide a cone of light when the condenser is in the high position. My
attention was called to this point by Dr. O. L. Thomas, who has been kind
enough to examine this new method of illumination.
One advantage of high-condenser illumination is that a perfectly structureless source of light need not be used, since the image of the light is not focused
in the plane of the object; further, there cannot be any interference with the
image by dust on any screen or cooling-chamber that may for any special
purpose be placed between the lamp and the condenser.
The direction and degree of obliquity of the light may also be controlled
by what I call 'low-condenser illumination', which is explained in the third
diagram of Text-fig. 1. The condenser is here placed below the 'critical'
position, and the source or light is thus focused below the object. An actual
image of the source of light is now produced just behind the back focal plane
of the objective, where it can be examined, if desired, by screwing a lowpower objective into the bottom of the draw-tube. This is not nearly such
an effective method for obtaining oblique illumination as that described above,
but it is useful in phase-contrast microscopy. If an annular stop be put in front
of the source of light, its image will be produced just behind the back focal
plane of the objective, where the phase-plate may conveniently be placed.
Dr. R. Barer has kindly read this paper and given me the benefit of his
criticism. He remarks that the method of illumination that I adopt is best
described by saying that the aperture-stop, usually situated just below the
condenser, has been removed to a considerable distance, so that the mirror
lies between the aperture-stop and the condenser. I thank Mr. I. C. J.
Galbraith, who gave valuable advice on an important theoretical point while
I was engaged in perfecting the method of oblique illumination described
in this paper.
SUMMARY
The purpose of the method is to facilitate the resolution of extremely fine
regularly-repeated markings on microscopical objects. Resolution is obtained
by using the objective as an eyepiece to observe the direction and degree of
obliquity of the light; these are controlled by movements of the mirror. To
enable the objective to be used in this way, the condenser is raised above the
238 Baker—A New Method for Oblique Microscopical Illumination
position required for so-called 'critical' illumination. The diffraction-spectrum formed by the markings on the object is clearly seen while the mirror
movements are being made, and it is easy to produce a bright and complete
spectrum.
REFERENCE
HARTRIDGE, H., 1919. Journ. Quekett micr. Club, 14, 73.